Somatostatin receptor 2 expression in nasopharyngeal cancer is induced by Epstein Barr virus infection: impact on prognosis, imaging and therapy.

Nasopharyngeal cancer (NPC), endemic in Southeast Asia, lacks effective diagnostic and therapeutic strategies. Even in high-income countries the 5-year survival rate for stage IV NPC is less than 40%. Here we report high somatostatin receptor 2 (SSTR2) expression in multiple clinical cohorts comprising 402 primary, locally recurrent and metastatic NPCs. We show that SSTR2 expression is induced by the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) via the NF-κB pathway. Using cell-based and preclinical rodent models, we demonstrate the therapeutic potential of SSTR2 targeting using a cytotoxic drug conjugate, PEN-221, which is found to be superior to FDA-approved SSTR2-binding cytostatic agents. Furthermore, we reveal significant correlation of SSTR expression with increased rates of survival and report in vivo uptake of the SSTR2-binding 68Ga-DOTA-peptide radioconjugate in PET-CT scanning in a clinical trial of NPC patients (NCT03670342). These findings reveal a key role in EBV-associated NPC for SSTR2 in infection, imaging, targeted therapy and survival.

Epigenetic landscape of pancreatic neuroendocrine tumours reveals distinct cells of origin and means of tumour progression.

Recent data suggest that Pancreatic Neuroendocrine Tumours (PanNETs) originate from α- or ß-cells of the islets of Langerhans. The majority of PanNETs are non-functional and do not express cell-type specific hormones. In the current study we examine whether tumour DNA methylation (DNAme) profiling combined with genomic data is able to identify cell of origin and to reveal pathways involved in PanNET progression. We analyse genome-wide DNAme data of 125 PanNETs and sorted α- and ß-cells. To confirm cell identity, we investigate ARX and PDX1 expression. Based on epigenetic similarities, PanNETs cluster in α-like, ß-like and intermediate tumours. The epigenetic similarity to α-cells progressively decreases in the intermediate tumours, which present unclear differentiation. Specific transcription factor methylation and expression vary in the respective α/ß-tumour groups. Depending on DNAme similarity to α/ß-cells, PanNETs have different mutational spectra, stage of the disease and prognosis, indicating potential means of PanNET progression.

Immune Surveillance in Clinical Regression of Preinvasive Squamous Cell Lung Cancer.

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.

The evolving (epi)genetic landscape of pancreatic neuroendocrine tumours.

Neuroendocrine neoplasms (NENs) are a relatively rare group of heterogeneous tumours originating from neuroendocrine cells found throughout the body. Pancreatic NENs (PanNENs) are the second most common pancreatic malignancy accounting for 1-3% of all neoplasms developing in the pancreas. Despite having a low background mutation rate, driver mutations in MEN1, DAXX/ATRX and mTOR pathway genes (PTEN, TSC1/2) are implicated in disease development and progression. Their increased incidence coupled with advances in sequencing technologies has reignited the interest in PanNEN research and has accelerated the acquisition of molecular data. Studies utilising such technological advances have further enriched our knowledge of PanNENs' biology through novel findings, including higher-than-expected presence of germline mutations in 17% of sporadic tumours of no familial background, identification of novel mutational signatures and complex chromosomal rearrangements and a dysregulated epigenetic machinery. Integrated genomic studies have progressed the field by identifying the synergistic action between different molecular mechanisms, while holding the promise for deciphering disease heterogeneity. Although our understanding is far from being complete, these novel findings have provided the optimism of shaping the future of PanNEN research, ultimately leading to an era of precision medicine for NETs. Here, we recapitulate the existing knowledge on pancreatic neuroendocrine tumours (PanNETs) and discuss how recent, novel findings have furthered our understanding of these complex tumours.

Deciphering the genomic, epigenomic, and transcriptomic landscapes of pre-invasive lung cancer lesions.

The molecular alterations that occur in cells before cancer is manifest are largely uncharted. Lung carcinoma in situ (CIS) lesions are the pre-invasive precursor to squamous cell carcinoma. Although microscopically identical, their future is in equipoise, with half progressing to invasive cancer and half regressing or remaining static. The cellular basis of this clinical observation is unknown. Here, we profile the genomic, transcriptomic, and epigenomic landscape of CIS in a unique patient cohort with longitudinally monitored pre-invasive disease. Predictive modeling identifies which lesions will progress with remarkable accuracy. We identify progression-specific methylation changes on a background of widespread heterogeneity, alongside a strong chromosomal instability signature. We observed mutations and copy number changes characteristic of cancer and chart their emergence, offering a window into early carcinogenesis. We anticipate that this new understanding of cancer precursor biology will improve early detection, reduce overtreatment, and foster preventative therapies targeting early clonal events in lung cancer.

Correlation of Smoking-Associated DNA Methylation Changes in Buccal Cells With DNA Methylation Changes in Epithelial Cancer.

IMPORTANCE: The utility of buccal cells as an epithelial source tissue for epigenome-wide association studies (EWASs) remains to be demonstrated. Given the direct exposure of buccal cells to potent carcinogens such as smoke, epigenetic changes in these cells may provide insights into the development of smoke-related cancers. OBJECTIVE: To perform an EWAS in buccal and blood cells to assess the relative effect of smoking on the DNA methylation (DNAme) patterns in these cell types and to test whether these DNAme changes are also seen in epithelial cancer. DESIGN, SETTING, AND PARTICIPANTS: In 2013, we measured DNAme at more than 480,000 CpG sites in buccal samples provided in 1999 by 790 women (all aged 53 years in 1999) from the United Kingdom Medical Research Council National Survey of Health and Development. This included matched blood samples from 152 women. We constructed a DNAme-based smoking index and tested its sensitivity and specificity to discriminate normal from cancer tissue in more than 5000 samples. MAIN OUTCOMES AND MEASURES: CpG sites whose DNAme level correlates with smoking pack-years, and construction of an associated sample-specific smoking index, which measures the mean deviation of DNAme at smoking-associated CpG sites from a normal reference. RESULTS: In a discovery set of 400 women, we identified 1501 smoking-associated CpG sites at a genome-wide significance level of P < 10-7, which were validated in an independent set of 390 women. This represented a 40-fold increase of differentially methylated sites in buccal cells compared with matched blood samples. Hypermethylated sites were enriched for bivalently marked genes and binding sites of transcription factors implicated in DNA repair and chromatin architecture (P < 10-10). A smoking index constructed from the DNAme changes in buccal cells was able to discriminate normal tissue from cancer tissue with a mean receiver operating characteristic area under the curve of 0.99 (range, 0.99-1.00) for lung cancers and of 0.91 (range, 0.71-1.00) for 13 other organs. The corresponding area under the curve of a smoking signature derived from blood cells was lower than that derived from buccal cells in 14 of 15 cancer types (Wilcoxon signed rank test, P = .001). CONCLUSIONS AND RELEVANCE: These data point toward buccal cells as being a more appropriate source of tissue than blood to conduct EWASs for smoking-related epithelial cancers.

Mobile DNA in cancer. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes.

Long interspersed nuclear element-1 (L1) retrotransposons are mobile repetitive elements that are abundant in the human genome. L1 elements propagate through RNA intermediates. In the germ line, neighboring, nonrepetitive sequences are occasionally mobilized by the L1 machinery, a process called 3' transduction. Because 3' transductions are potentially mutagenic, we explored the extent to which they occur somatically during tumorigenesis. Studying cancer genomes from 244 patients, we found that tumors from 53% of the patients had somatic retrotranspositions, of which 24% were 3' transductions. Fingerprinting of donor L1s revealed that a handful of source L1 elements in a tumor can spawn from tens to hundreds of 3' transductions, which can themselves seed further retrotranspositions. The activity of individual L1 elements fluctuated during tumor evolution and correlated with L1 promoter hypomethylation. The 3' transductions disseminated genes, exons, and regulatory elements to new locations, most often to heterochromatic regions of the genome.

Processed pseudogenes acquired somatically during cancer development.

Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5' truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context.

Cell migration leads to spatially distinct but clonally related airway cancer precursors.

BACKGROUND: Squamous cell carcinoma of the lung is a common cancer with 95% mortality at 5 years. These cancers arise from preinvasive lesions, which have a natural history of development progressing through increasing severity of dysplasia to carcinoma in situ (CIS), and in some cases, ending in transformation to invasive carcinoma. Synchronous preinvasive lesions identified at autopsy have been previously shown to be clonally related. METHODS: Using autofluorescence bronchoscopy that allows visual observation of preinvasive lesions within the upper airways, together with molecular profiling of biopsies using gene sequencing and loss-of-heterozygosity analysis from both preinvasive lesions and from intervening normal tissue, we have monitored individual lesions longitudinally and documented their visual, histological and molecular relationship. RESULTS: We demonstrate that rather than forming a contiguous field of abnormal tissue, clonal CIS lesions can develop at multiple anatomically discrete sites over time. Further, we demonstrate that patients with CIS in the trachea have invariably had previous lesions that have migrated proximally, and in one case, into the other lung over a period of 12 years. CONCLUSIONS: Molecular information from these unique biopsies provides for the first time evidence that field cancerisation of the upper airways can occur through cell migration rather than via local contiguous cellular expansion as previously thought. Our findings urge a clinical strategy of ablating high-grade premalignant airway lesions with subsequent attentive surveillance for recurrence in the bronchial tree.

Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors.

Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to "neutral drift" of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease. DOI:http://dx.doi.org/10.7554/eLife.00966.001.

Genomic evidence of pre-invasive clonal expansion, dispersal and progression in bronchial dysplasia.

The term 'field cancerization' is used to describe an epithelial surface that has a propensity to develop cancerous lesions, and in the case of the aerodigestive tract this is often as a result of chronic exposure to carcinogens in cigarette smoke 1, 2. The clinical endpoint is the development of multiple tumours, either simultaneously or sequentially in the same epithelial surface. The mechanisms underlying this process remain unclear; one possible explanation is that the epithelium is colonized by a clonal population of cells that are at increased risk of progression to cancer. We now address this possibility in a short case series, using individual genomic events as molecular biomarkers of clonality. In squamous lung cancer the most common genomic aberration is 3q amplification. We use a digital PCR technique to assess the clonal relationships between multiple biopsies in a longitudinal bronchoscopic study, using amplicon boundaries as markers of clonality. We demonstrate that clonality can readily be defined by these analyses and confirm that field cancerization occurs at a pre-invasive stage and that pre-invasive lesions and subsequent cancers are clonally related. We show that while the amplicon boundaries can be shared between different biopsies, the degree of 3q amplification and the internal structure of the 3q amplicon varies from lesion to lesion. Finally, in this small cohort, the degree of 3q amplification corresponds to clinical progression.

HIF-1alpha mRNA gene expression levels in improved diagnosis of early stages of prostate cancer.

OBJECTIVES: Several clinical studies have indicated that the rates of invasive growth and metastatic disease in cancer depend on the degree of hypoxia, which is mediated by hypoxia-inducible factor 1 alpha (HIF-1alpha). To determine its potential role as a marker for prostate cancer (CaP) diagnosis, HIF-1alpha mRNA levels were measured in blood samples of patients diagnosed with different stages of prostatic disease. METHODS: HIF-1alpha mRNA levels were measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and correlated with accurate clinicopathological data. Quantitative data were compared with serum prostate-specific antigen (PSA) measurements to determine variations in the accuracy of the CaP diagnosis. RESULTS: HIF-1alpha mRNA levels were significantly upregulated in patients with localized CaP (LocCaP; n=63; p<0.0001), compared with patients with no evidence of malignancy (NEOM) and benign prostatic hyperplasia (BPH) (n=35 for both patient groups combined). Receiver operator characteristic (ROC) curve analysis demonstrated that HIF-1alpha specificity for the NEOM/BPH diagnosis was 88.6%. Sensitivity for LocCaP was 74.6% with an overall diagnostic efficiency of 79.6%. Specificity of the NEOM diagnosis at PSA levels of 4.0 ng ml(-1) was 28.6% and sensitivity of the LocCap diagnosis was 65.1%, demonstrating a reduced overall diagnostic efficiency, compared with that given by HIF-1alpha measurements, of 52.0%. Levels of HIF-1alpha in patients with metastatic CaP (MetCaP; n=27)) were similar to those in the NEOM/BPH group. CONCLUSIONS: HIF-1alpha is upregulated early in CaP development with subsequent downregulation at later metastatic stages. This study demonstrates increased accuracy of early-stage disease diagnosis using HIF-1alpha qRT-PCR compared with serum PSA measurements. HIF-1alpha may therefore be a useful adjunct, together with other diagnostic markers used in relative qRT-PCR and current diagnostic techniques (including serum PSA and PSA velocity) to minimize unnecessary biopsies indicated by elevated serum PSA levels alone.

Measurement of blood E2F3 mRNA in prostate cancer by quantitative RT-PCR: a preliminary study.

The use of serum prostate-specific antigen (PSA) measurements necessitates biopsies for accurate prostate cancer (CaP) diagnosis. Overall efficiency of accurate diagnosis, when PSA levels are used alone, is less than 60%. E2F3 was evaluated as an alternative biomarker using patient blood samples. Expression levels were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and correlated with accurate clinicopathological data. Statistical analysis demonstrated significant differences in E2F3 expression levels (p<0.0001), and high levels of discrimination (receiver operator curve/area under curve analysis values (AUC) >0.88), in particular at early stages of disease development, between benign disease and localized CaP. Limited levels of discrimination were observed at the later stages of disease development, between localized and metastatic disease (p=0.076, AUC=0.633). A cut-off point of 0.34 with high specificity for benign disease (92.3%) and sensitivity for CaP diagnosis (81.0%) was identified. At this cut-off point, 85% patients were correctly diagnosed with either malignant or benign disease. This study demonstrates the strength of E2F3 as a potential marker for discriminating benign and malignant disease, addressing the current limitations of serum PSA measurements.